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Biomedical subjects

T Takeuchi

Publications and source records attributed to T Takeuchi.

At least 235 records · Page 13Linked to original sources
PubMed2000

[A case of traumatic priapism].

We report a case of high flow priapism secondary to arteriovenous fistula produced by perineal trauma in a 43-year-old patient. Diagnosis was based on the result of gasometry in cavernous blood and color doppler sonography. Superselective embolization of the deep artery of the penis was performed with good results. After 6 months of treatment, erection and sexual function were normal. Our case demonstrates that this procedure is a safe and effective therapy for high flow priapism.

Adult↗
PubMed1999

Temporal cortex activation during speech recognition: an optical topography study.

Cortical activity during speech recognition was examined using optical topography (OT), a recently developed non-invasive technique. To assess relative changes in hemoglobin oxygenation, local changes in near-infrared light absorption were measured simultaneously from 44 points in both hemispheres. A dichotic listening paradigm was used in this experiment, in which target stimuli and non-target stimuli were presented to different ears. Subjects were asked to track targets and to press a button when targets shifted from one ear to the other. We compared three tasks: (i) a control task, in which a tone was used as the target; (ii) a repeat task, in which the target was one repeated sentence; (iii) a story task, in which the targets were continuous sentences of a story. The activity for the story task, compared with the repeat task, was localized in the left superior temporal cortex. Relative to the control task, we observed in this region a larger increase in oxyhemoglobin concentration and a decrease in deoxyhemoglobin concentration in the story task than those in the repeat task. These results suggest that the activity in the left temporal association area reflects the load of auditory, memory, and language information processing.

Adult↗
PubMed1999

Separation of enantiomers on anion exchangers modified with heparin in liquid chromatography.

The separation of enantiomers of chloroquine and other amine derivatives has been investigated by liquid chromatography using anion exchangers modified with heparin as the stationary phase. Modification and mobile phase conditions affected the separation of analytes. Separation of the chloroquine enantiomers could be achieved by using a buffer solution containing acetonitrile as the mobile phase.

Acetonitriles↗
PubMed1999

Cytostatin, an inhibitor of cell adhesion to extracellular matrix, selectively inhibits protein phosphatase 2A.

Cytostatin, which is isolated from a microbial cultured broth as a low molecular weight inhibitor of cell adhesion to extracellular matrix (ECM), has anti-metastatic activity against B16 melanoma cells in vivo. In this study, we examined a target of cytostatin inhibiting cell adhesion to ECM. Cytostatin inhibited tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin upon B16 cell adhesion to fibronectin. While the amount of FAK was not affected by cytostatin, electrophoretically slow-migrating paxillin appeared. Alkaline phosphatase treatment diminished cytostatin-induced slow-migrating paxillin. Furthermore, cytostatin increased intracellular serine/threonine-phosphorylated proteins and was found to be a selective inhibitor of protein phosphatase 2A (PP2A). Cytostatin inhibited PP2A with an IC(50) of 0.09 microgram/ml in a non-competitive manner against a substrate, p-nitrophenyl phosphate, but it had no apparent effect on other protein phosphatases including PP1, PP2B and alkaline phosphatase even at 100 microgram/ml. On the contrary, dephosphocytostatin, a cytostatin analogue, without inhibitory effect on PP2A did not affect B16 cell adhesion including FAK and paxillin. These results indicate that cytostatin inhibits cell adhesion through modification of focal contact proteins such as paxillin by inhibiting a PP2A type protein serine/threonine phosphatase. This is the first report that describes a drug with anti-metastatic ability that inhibits PP2A selectively.

Animals↗
PubMed1999

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

Acetyltransferases↗
PubMed1999

Novel rabphilin-3-like protein associates with insulin-containing granules in pancreatic beta cells.

A novel rabphilin-3-like gene, granuphilin, has been identified in pancreatic beta cells by comparing genes expressed in pancreatic alpha and beta cell lines using mRNA differential display. The domain structure of the protein products of the granuphilin gene contains an amino-terminal zinc-finger motif and carboxyl-terminal C(2)-domains, similar to that of the rabphilin-3 gene. There are two isoforms: the larger isoform, granuphilin-a, has two C(2)-domains, whereas the smaller one, granuphilin-b, contains only the first C(2)-domain. Granuphilin is specifically expressed in pancreatic beta cells and the pituitary gland, but not in pancreatic alpha cells, the adrenal gland, or other major organs such as the brain. A portion of granuphilin associates with insulin-containing dense-core granules, but not with synaptic-like microvesicles in beta cells. Thus, its distribution pattern presents a striking contrast with that of rabphilin-3, which associates with small synaptic vesicles in neurons. The first C(2)-domain of granuphilin binds phospholipids in a Ca(2+)-independent manner, whereas the second one does not. These distinctive characteristics of granuphilin suggest that it is not a simple counterpart of rabphilin-3 in endocrine cells and that it has a unique role in the regulated exocytosis of dense-core granules in endocrine tissues.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue.

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a gene encoding phosphatidyl inositol-specific phospholipase C from Trypanosoma cruzi.

A gene encoding phosphatidyl inositol-4,5-bisphosphate phospholipase C (PLC) was cloned from the protozoan parasite Trypanosoma cruzi. A partial cDNA encoding putative PLC was obtained by a polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved regions of PLCs. A 2178-bp protein coding region of the T. cruzi PLC gene, composed from cDNA and genomic clones, encodes a putative PLC with a calculated molecular mass of 82,032 Da and an isoelectric point of 5.93. The deduced amino acid sequence of T. cruzi PLC exhibited 23-42% overall identities with the PLCs from other organisms. Among them, PLC from Ictalurus punctatus revealed the highest identity to T. cruzi PLC. The percentage identities of the entire proteins and the catalytic X/Y domains suggested that T. cruzi PLC is more evolutionarily related to the PLCs of higher eukaryotes than to those of lower unicellular eukaryotes. The tetrad analysis of the segregants of the Saccharomyces cerevisiae PLC1/plc1::HIS3 diploid strain transformed with the T. cruzi PLC-expressing plasmid showed that expression of T. cruzi PLC suppressed the growth defect caused by the plc1 disruption in yeasts. Temperature-sensitive phenotype of the S. cerevisiae plc1-mutant haploid strain was also suppressed by the expression of T. cruzi PLC. The phosphatidyl inositol-4,5-biphosphate (PtdIns(4,5)P2) hydrolyzing activity of T. cruzi PLC was demonstrated in the lysate from the plc1-temperature sensitive yeast mutant strain transformed with the T. cruzi PLC-expressing plasmid. The yeast-expressed T. cruzi PLC showed an absolute Ca2+ dependence which was similar to mammalian PLC isoforms: the half-maximal activity at 0.5-1 x 10(-5) M Ca2+ and the maximal activity at 1-2 x 10(-4) M Ca2+.

Amino Acid Sequence↗
PubMed1999

Preferential localization of CD8+ alpha E beta 7+ T cells around acinar epithelial cells with apoptosis in patients with Sjögren's syndrome.

The T lymphocytes that infiltrate the exocrine glands in Sjögren's syndrome (SS) play a key role in damaging glandular epithelial cells, but the mechanisms of this damage by T lymphocytes are not fully understood. To determine the cellular basis of this phenomenon, we focused our attention on the T lymphocytes around acinar epithelial cells in SS. We showed that CD8+ but not CD4+ T lymphocytes were located around the acinar epithelial cells and that a majority of these CD8+ T lymphocytes possess an unique integrin, alpha E beta 7 (CD103). The acinar epithelial cell adherent with alpha E beta 7 (CD103)+ CD8+ T lymphocytes was apoptotic. Both the perforin/granzyme B and Fas/Fas ligand pathways were implicated in the process of programmed cell death in lacrimal glands. These results suggested that alpha E beta 7 integrin, by interacting with E-cadherin, mediates the adhesion between CD8+ T lymphocytes and acinar epithelial cells in SS and participates in inducing epithelial cell apoptosis, leading to secretory dysfunction of exocrine glands, a hallmark of SS.

Adult↗
PubMed1999

A new human synovial sarcoma cell line, HS-SY-3, with a truncated form of hybrid SYT/SSX1 gene.

Recent cytogenetical and molecular studies have indicated that synovial sarcoma harbors a t(X;18)(p11.2;q11.2) translocation, resulting in the formation of a hybrid SYT/SSX (SSX1 or SSX2) gene. We newly established a human cell line, HS-SY-3, from a synovial sarcoma. HS-SY-3 cells were shown to harbor the pathognomonic t(X;18)(p11.2;q11.2) translocation by chromosome analysis but not to exhibit the classical hybrid SYT/SSX transcripts induced by this translocation, using RT-PCR. To determine the reason for this discrepancy, we analyzed cDNA from HS-SY-3 cells, as well as the original sarcoma tissue by the rapid amplification of cDNA 3' end assay, and found that the chimaeric cDNA was 240 bp shorter than the previously established SYT/SSX1 cDNA due to truncation of the 3' side of SSX1. The HS-SY-3 cells should be useful for future functional studies of the SYT/SSX chimeric gene.

Aged↗
PubMed1999

Effect of eluent composition on retention behavior of anions in ion chromatography on anion-exchangers modified with heparin.

Effect of eluent composition on retention behavior of inorganic anions have been investigated in ion chromatography using anion-exchangers modified with heparin. Both cation and anion of the eluent affected the retention of analyte anions and unusual retention behavior was observed on the modified stationary phase. The retention time of anions decreased with decreasing eluent concentration when sodium sulfate, magnesium sulfate and chlorides of alkali metals were used as the eluent, whereas it increased with decreasing eluent concentration when aluminum sulfate, copper sulfate and sulfuric acid were used as the eluent. The retention of nitrate increased in the order of Li+, Na+, K+, Rb+ and Cs+ when their chlorides were used as the eluent. When sodium perchlorate and chlorides of alkaline-earth metals were used as the eluent, the eluent should include heparin. Otherwise, the modifier was partially bled from the column.

Anions↗
PubMed1999

Biomechanical role of the intervertebral disc and costovertebral joint in stability of the thoracic spine. A canine model study.

STUDY DESIGN: Biomechanical evaluation was performed to investigate the stability of the thoracic spine. Unilateral resection of the intervertebral disc, the rib head joint, and the costotransverse joint were sequentially performed, and nondestructive cyclic loading tests were conducted at each injury stage to examine the roles of the intervertebral disc and the costovertebral joint of the thoracic spine. The effects of each resection were three-dimensionally analyzed as the main motion and the associated coupled motions. OBJECTIVE: To examine the role of the intervertebral disc and the costovertebral joint in stability of the thoracic spine. SUMMARY OF BACKGROUND DATA: The effects of unilateral resection of the intervertebral disc and the costovertebral joints in the thoracic spine with the rib cage have not been documented three-dimensionally in a biomechanical study. MATERIALS AND METHODS: Ten canine rib cage-thoracic spine complexes, consisting of the sixth to eighth ribs, the sternum and T5-T8 vertebrae, were used. Six pure moments along three axes, flexion-extension, lateral bending, and axial rotation, were applied to the specimen, and the angular deformation between T6-T7 was recorded by a stereophotogrammetric method. After the intact specimens were tested, staged resections were conducted in the following manner: partial resection of the T6-T7 intervertebral disc, performed as a resection of the anterior longitudinal ligament, the nucleus pulposus, and the annulus fibrosus on the approach side, leaving the posterior longitudinal ligament intact; resection of the right seventh rib head with the joint capsule; and resection of the right seventh costotransverse joint. At each stage, the main motion and associated coupled motions were determined three dimensionally. RESULTS: The ranges of motion (ROM) in flexion-extension, lateral bending, and axial rotation were significantly increased after partial discectomy (P < 0.01). Moreover, along with large increases in the ROM of the main motions in left axial rotation and right lateral bending, coupled motions, expressed by right lateral bending and left axial rotation, showed marked increases after resection of the rib head joint (P < 0.05). The neutral zones also increased in lateral bending, axial rotation, and flexion-extension after partial discectomy (P < 0.01). A further increase in the neutral zone was observed in lateral bending after resection of the right seventh rib head (P < 0.01). CONCLUSIONS: In this canine spine model, the intervertebral disc regulates the stability of the thoracic spine in flexion-extension, lateral bending, and axial rotation. Moreover, the articulation of the rib head with the vertebral bodies provides stability to the thoracic spine in lateral bending and axial rotation. Unilateral resection of the rib head joint after partial discectomy on the same side produces significant coupled motions in lateral bending and axial rotation, resulting in a significant decrease in thoracic spinal stability, and integrity.

Animals↗
PubMed1999

Increased anxiety and impaired pain response in puromycin-sensitive aminopeptidase gene-deficient mice obtained by a mouse gene-trap method.

A mouse mutation, termed goku, was generated by a gene-trap strategy. goku homozygous mice showed dwarfism, a marked increase in anxiety, and an analgesic effect. Molecular analysis indicated that the mutated gene encodes a puromycin-sensitive aminopeptidase (Psa; EC 3. 4.11.14), whose functions in vivo are unknown. Transcriptional arrest of the Psa gene and a drastic decrease of aminopeptidase activity indicated that the function of Psa is disrupted in homozygous mice. Together with the finding that the Psa gene is strongly expressed in the brain, especially in the striatum and hippocampus, these results suggest that the Psa gene is required for normal growth and the behavior associated with anxiety and pain.

Aminopeptidases↗
PubMed1999

Critical role of sulfenic acid formation of thiols in the inactivation of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide.

The relationship between possible modifications of the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) and modified enzyme activity was examined. There are 16 free thiols, including 4 active site thiols, in a tetramer of GAPDH molecule. NO donors, sodium nitroprusside (SNP), and S-nitroso-N-acetyl-DL-penicillamine (SNAP) decreased the number of free thiols with a concomitant inhibition of GAPDH activity in a concentration- and time-dependent manner. After treatment for 30 min, free thiols were maximally decreased to 8-10 per GAPDH tetramer and enzyme activity was also inhibited to 5-10% of control activity. In the presence of 30 mM dithiothreitol (DTT), these effects were completely blocked. Since similar results were obtained in the case of hydrogen peroxide (H2O2) treatment, which is known to oxidize the thiols, these effects of nitric oxide donors were probably due to modification of thiol groups present in a GAPDH molecule. On the other hand, DTT posttreatment after the treatment of GAPDH with SNP, SNAP, or H2O2 did not completely restore the modified thiols and the inhibited enzyme activity. DTT posttreatment after the 30-min-treatment with these agents restored free thiols to 14 in all treatments. In the case of SNAP treatment, all 4 active sites were restored and enzyme activity reached more than 80% of the control activity, but in two other cases one active site remained modified and enzyme activity was restored to about only 20%. Therefore, all 4 free thiols in the active site seem to be very important for full enzyme activity. DTT posttreatment in the presence of sodium arsenite, which is known to reduce sulfenic acid to thiol, almost completely restored both thiol groups and enzyme activity. These findings suggest that nitric oxide inhibits GAPDH activity by modifications of the thiols which are essential for this activity, and that the modification includes formation of sulfenic acid, which is not restored by DTT. S-nitrosylation, which is one type of thiol modification by NO, occurred when GAPDH was treated with SNAP but not SNP. Analysis of thiol modification showed that SNAP preferentially nitrosylated the active site thiols, the nitrosylation of which fully disappeared by DTT posttreatment. It seems that SNAP nitrosylates the active site thiols of GAPDH to prevent these thiols from oxidizing to sulfenic acid.

Binding Sites↗
PubMed1999

Separation and sensing based on molecular recognition using molecularly imprinted polymers.

Molecular recognition-based separation and sensing systems have received much attention in various fields because of their high selectivity for target molecules. Molecular imprinting has been recognized as a promising technique for the development of such systems, where the molecule to be recognized is added to a reaction mixture of a cross-linker(s), a solvent(s), and a functional monomer(s) that possesses a functional groups(s) capable of interacting with the target molecule. Binding sites in the resultant polymers involve functional groups originating from the added functional monomer(s), which can be constructed according to the shape and chemical properties of the target molecules. After removal of the target molecules, these molecularly imprinted complementary binding sites exhibit high selectivity and affinity for the template molecule. In this article, recent developments in molecularly imprinted polymers are described with their applications as separation media in liquid chromatography, capillary electrophoresis, solid-phase extraction, and membranes. Examples of binding assays and sensing systems using molecularly imprinted polymers are also presented.

Chemistry Techniques, Analytical↗
PubMed1999

Induction of oxidative DNA damage in anaerobes.

We compared oxidative DNA damage in strictly anaerobic Prevotella melaninogenica, aerotolerant anaerobic Bacteroides fragilis, and facultative anaerobic Salmonella typhimurium after exposure to O2 or H2O2. Using HPLC with electrochemical detection, we measured 8-hydroxydeoxyguanosine (8OHdG) as a damage marker. O2 induced 8OHdG in P. melaninogenica but not in B. fragilis, which shows catalase activity, or in S. typhimurium. In P. melaninogenica, with catalase, O2 induced less 8OHdG; superoxide dismutase had no effect; with glucose and glucose oxidase, O2 induced more 8OHdG. H2O2 also markedly increased 8OHdG. O2 was suggested to induce 8OHdG through H2O2. O2 or H2O2 decreased survival only in P. melaninogenica. Highly sensitive to oxidative stress, P. melaninogenica could prove useful for investigating oxidative DNA damage.

Anaerobiosis↗